Abstract
Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.
Highlights
The membrane-embedded complexes driving oxidative phosphorylation in mitochondria consist of subunits that are encoded in either the nuclear or the organellar DNA
One region of the ribosome that is of special importance for the fate of newly synthesized polypeptides is the ribosomal tunnel exit where nascent chains are exposed to a hydrophilic milieu for the first time
We concluded that the Strategy to Identify Proteins Located at the Mitochondrial addition of the C-terminal His7 tag impaired the function of Ribosomal Tunnel Exit—The rim of the tunnel exit of bacterial Mrpl22 and continued our analysis only with ribosomes is composed of the conserved proteins L22, L23, L24, the fully functional proteins Mrpl4His7, Mrp20His7, and and L29 (Fig. 1A)
Summary
To identify proteins located in proximity to the mitochondrial ribosomal tunnel exit, we mapped the vicinity of the tunnel exit proteins using crosslinking and characterization of the purified cross-linking products via mass spectrometry. We concluded that the Strategy to Identify Proteins Located at the Mitochondrial addition of the C-terminal His7 tag impaired the function of Ribosomal Tunnel Exit—The rim of the tunnel exit of bacterial Mrpl22 and continued our analysis only with ribosomes is composed of the conserved proteins L22, L23, L24, the fully functional proteins Mrpl4His7, Mrp20His7, and and L29 (Fig. 1A).
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